1. Field of the Invention
The present invention relates to novel peptidomimetics and other compounds which are useful as inhibitors of protein isoprenyl transferases (particularly protein farnesyltransferase and geranylgeranyltransferase) and as anti-cancer drugs, to compositions containing such compounds and to methods of use.
2. Background Information
Ras proteins are plasma membrane-associated GTPases that function as relay switches that transduce biological information from extracellular signals to the nucleus (29-31). In normal cells Ras proteins cycle between the GDP-(inactive) and GTP-(active) bound forms to regulate proliferation and differentiation. The mechanism by which extracellular signals, such as epidermal and platelet derived growth factor (EGF and PDGF), transduce their biological information to the nucleus via Ras proteins has recently been unraveled (29-31). Binding of the growth factors to tyrosine kinase receptors results in autophosphorylation of various tyrosines which then bind src-homology 2 (SH2) domains of several signaling proteins. One of these, a cytosolic complex of GRB-2 and a ras exchanger (m-SOS-1), is recruited by the tyrosine phosphorylated receptor where mSOS-1 catalyzes the exchange of GDP for GTP on Ras, hence activating it. GTP-bound Ras recruits Raf, a serine/threonine kinase, to the plasma membrane where it is activated. Raf triggers a kinase cascade by phosphorylating mitogen-activated protein (MAP) kinase/extracellular-regulated protein kinase (ERK) kinase (MEK) which in turn phosphorylates MAP Kinase on threonine and tyrosine residues. Activated MAP Kinase translocates to the nucleus where it phosphorylates transcription factors (31). Termination of this growth signal is accomplished by hydrolysis of Ras-GTP to Ras-GDP.
Ras oncogenes are the most frequently identified activated oncogenes in human tumors (1-3). In a large number of human cancers, Ras is GTP-locked because of mutations in amino acids 12, 13, or 61 and the above Ras pathway no longer requires an upstream growth signal and is uninterrupted. As a consequence, enzymes in this pathway such as Raf, MEK and MAP Kinase are constitutively activated.
In addition to its inability to hydrolyze GTP, oncogenic Ras must be plasma membrane-bound to cause malignant transformation (13). Ras is posttranslationally modified by a lipid group, farnesyl, which mediates its association with the plasma membrane (10-14).
Post-translational events leading to membrane association of p21ras have previously been disclosed (10-14). The p21ras proteins are first made as pro-p21ras in the cytosol where they are modified on cysteine 186 of their carboxyl terminal sequence CA.sub.1 A.sub.2 X (C=cysteine, A.sub.1 and A.sub.2 =isoleucine, leucine or valine and X=methionine or serine) by the cholesterol biosynthesis intermediate farnesyl pyrophosphate (FPP). This farnesylation reaction is then followed by peptidase removal of the A.sub.1 A.sub.2 X tripeptide and carboxymethylation of the remaining cysteine. The processed p21ras proteins associate with the inner surface of the plasma membrane (10-14).
p21Ras farnesyltransferase, the enzyme responsible for catalyzing the transfer of farnesyl, a 15-carbon isoprenoid, from FPP to the cysteine of the CA.sub.1 A.sub.2 X carboxyl terminus of p21ras, has been purified to homogeneity from rat brain (15,16). The enzyme is a heterodimer composed of .alpha. and .beta. subunits of molecular weights 49 and 46 kDa, respectively (17). The .beta. subunit has been shown to bind p21ras (17). Because p21ras farnesylation and subsequent membrane association are required for p21ras transforming activity (13), it has been proposed that p21ras farnesyltransferase would be a useful anticancer therapy target. Accordingly, an intensive search for inhibitors of the enzyme is underway (18-24, 33-44). Potential inhibitor candidates are CA.sub.1 A.sub.2 X tetrapeptides which have been shown to be farnesylated by p21ras farnesyltransferase and appear to be potent inhibitors of this enzyme in vitro (15,18,21-24). Competition studies have demonstrated that CA.sub.1 A.sub.2 X peptides with the greatest inhibitory activity are those where A.sub.1 and A.sub.2 are hydrophobic peptides with charged or hydrophilic residues in the central positions demonstrating very little inhibitory activity (18,21,23). A major drawback with the use of peptides as therapeutic agents is their low cellular uptake and their rapid inactivation by proteases.
The research efforts directed towards farnesyltransferase and the inhibition of its activity are further illustrated by the following patents or published patent applications:
U.S. Pat. No. 5,141,851 PA1 WO 91/16340 PA1 WO 92/18465 PA1 EPA 0456180 A1 PA1 EPA 0461869 A2 PA1 EPA 0512865 A2 PA1 EPA 0520823 A2 PA1 EPA 0523873 A1 PA1 Cys-Val-Leu-Ser PA1 Cys-Val-Ile-Met PA1 Cys-Val-Val-Met PA1 ii) lower alkyl; PA1 iii) alkenyl; PA1 iv) alkoxy; PA1 v) thioalkoxy; PA1 vi) halo; PA1 vii) haloalkyl; PA1 viii) aryl-L.sub.2 --, wherein L.sub.2 is absent, --CH.sub.2 --, --CH.sub.2 CH.sub.2 --, --CH(CH.sub.3)--, --O--, --S(O).sub.q wherein q is 0, 1, or 2, --N(R')-- wherein R' is hydrogen or lower alkyl, or --C(O)-- and aryl is selected from the group consisting of phenyl, naphthyl, tetrahydronaphthyl, indanyl and indenyl and the aryl group is unsubstituted or substituted; or PA1 ix) heterocyclic-L.sub.3 -- wherein L.sub.3 is absent, --CH.sub.2 --, --CH.sub.2 CH.sub.2 --, --CH(CH.sub.3)--, --O--, --S(O).sub.q wherein q is 0, 1 or 2, --N(R')-- wherein R' is hydrogen or loweralkyl, or --C(O)-- and heterocyclic is a monocyclic heterocyclic wherein the heterocyclic is unsubstituted or substituted with one, two, or three substituents independently selected from the group consisting of loweralkyl, hydroxy, hydroxyalkyl, halo, nitro, oxo (.dbd.O), amino, N-protected amino, alkoxy, thioalkoxy and haloalkyl; PA1 i) ##STR2## wherein R.sub.12a is hydrogen, loweralkyl or --C(O)O--R.sub.13, wherein R.sub.13 is hydrogen or a carboxy-protecting group and R.sub.12b is hydrogen or loweralkyl, with the proviso that R.sub.12a and R.sub.12b are not both hydrogen, PA1 ii) --C(O)NH--CH(R.sub.14)--C(O)OR.sub.15 wherein R.sub.14 is PA1 a) loweralkyl, PA1 b) cycloalkyl, PA1 c) cycloalkylalkyl, PA1 d) alkoxyalkyl, PA1 e) thioalkoxyalkyl, PA1 f) hydroxyalkyl, PA1 g) aminoalkyl, PA1 h) carboxyalkyl, PA1 i) alkoxycarbonylalkyl, PA1 j) arylalkyl or PA1 k) alkylsulfonylalkyl and PA1 iii) ##STR3## PA1 a) hydroxy, PA1 b) --SH, PA1 c) halo, PA1 d) oxo (.dbd.O), PA1 e) thioxo (.dbd.S), PA1 f) amino, PA1 g) --NHOH, PA1 h) alkylamino, PA1 i) dialkylamino, PA1 j) alkoxy, PA1 k) alkoxyalkoxy. PA1 l) haloalkyl. PA1 m) hydroxyalkyl, PA1 n) alkoxyalkyl, PA1 o) cycloalkyl, PA1 p) cycloalkenyl, PA1 q) alkenyl, PA1 r) alkynyl, PA1 s) aryl, PA1 t) arylalkyl, PA1 u) --COOH, PA1 v) --SO.sub.3 H, PA1 w) loweralkyl, PA1 x) alkoxycarbonyl, PA1 y) --C(O)NH.sub.2, PA1 z) --C(S)NH.sub.2, PA1 aa) --C(.dbd.N--OH)NH.sub.2, PA1 bb) loweralkyl-C(O)--, PA1 cc) loweralkyl-C(S)--, PA1 dd) formyl, PA1 ee) cyano, and PA1 ff) nitro.
Of the above disclosures, EPA 0520823 A2 discloses compounds which are useful in the inhibition of farnesyl-protein transferase and the farnesylation of the oncogene protein ras. The compounds of EPA 0520823 A2 are illustrated by the formula: EQU Cys-Xaa.sup.1 -dXaa.sup.2 -Xaa.sup.3
or pharmaceutically acceptable salts thereof, wherein Cys is a cysteine amino acid;
Xaa.sup.1 is an amino acid in natural L-isomer form;
dXaa2 is an amino acid in unnatural D-isomer form; and
Xaa.sup.3 is an amino acid in natural L-isomer form.
The preferred compounds are said to be CV(Dl)S and CV(Df)M, the amino acids being identified by conventional 3 letter and single letter abbreviations as follows:
______________________________________ Cysteine Cys C Glycine Gly G Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Serine Ser S Threonine Thr T Valine Val V ______________________________________
EPA 0523873 A1 discloses a modification of the compounds of EPA 0520823 A2 wherein Xaa.sup.3 is phenylalanine or p-fluorophenylalanine.
EPA 0461869 describes compounds which inhibit farnesylation of Ras protein of the formula: EQU Cys-Aaa.sup.1 -Aaa.sup.2 -Xaa
where Aaa.sup.1 and Aaa.sup.2 are aliphatic amino acids and Xaa is an amino acid. The aliphatic amino acids which are disclosed are Ala, Val, Leu and Ile. Preferred compounds are those where Aaa.sup.1 is Val, Aaa.sup.2 is Leu, Ile or Val and Xaa is Ser or Met. Preferred specific compounds are:
U.S. Pat. No. 5,141,851 and WO 91/16340 disclose the purified farnesyl protein transferase and certain peptide inhibitors therefor, including, for example, CVIM, TKCVIM and KKSKTKCVIM.
WO 92/18465 discloses certain farnesyl compounds which inhibit the enzymatic methylation of proteins including ras proteins.
EPA 0456180 A1 is directed to a farnesylprotein transferase assay which can be used to identify substances that block farnesylation of ras oncogene gene products while EPA 0512865 A2 discloses certain cyclic compounds that are useful for lowering cholesterol and inhibiting farnesylprotein transferase.
As will be evident from the foregoing, there is a great deal of research effort directed towards the development of inhibitors of farnesyltransferase. However, there still remains a need for improvements in this critically important area.
An enzyme closely related to farnesyltransferase, geranylgeranyltransferase I (GGTase I), attaches the lipid geranylgeranyl to the cysteine of the CAAX box of proteins where X is leucine (49,69). FTase and GGTase I are .alpha./.beta. heterodimers that share the .alpha. subunit (61,62). Cross-linking experiments suggested that both substrates (FPP and Ras CAAX) interact with the .beta. submit of FTase (17,63). Although GGTase I prefers leucine at the X position, its substrate specificity was shown to overlap with that of FTase in vitro (64). Furthermore, GGTase I is also able to transfer farnesyl to a leucine terminating peptide (65).
Although CAAX peptides are potent competitive inhibitors of FTase, rapid degradation and low cellular uptake limit their use as therapeutic agents. The stragegy of the present invention to develop superior compounds for inhibiting FTase and GGTase has been to replace several amino acids in the CAAX motif by peptidemimics. The rationale behind this strategy is based on the existance of a hydrophobic pocket at the enzyme active site that interacts with the hydrophobic "AA" dipeptide of the carboxyl termini CAAX of Ras molecules. In this regard, two very potent inhibitors of FTase (i.e. Cys-3AMBA-Met and Cys-4ABA-Met) were disclosed by us in an earlier U.S. patent application. The peptidomimetic Cys-4ABA-Met incorporates a hydrophobic/aromatic spacer (i.e. 4-aminobenzoic acid) between Cys and Met. The present application discloses several derivatives of Cys-4ABA-Met where positions 2 and 3 of 4-amino benzoic acid were modified by several alkyl, and/or aromatic groups, compounds that show great promise of ability to selectively antagonize RAS-dependent signaling and to selectively inhibit the growth of human tumors with aberrant Ras function.
Of the four types of Ras proteins (H--, N--, K4A--, and K4B-Ras) expressed by mammalian cells, K4B-Ras (also called K-Ras4B) is the most frequently mutated form of Ras in human cancers (1,3). Although several laboratories have demonstrated potent inhibition of oncogenic H-Ras processing and signaling (43,44), this disruption has not been shown with K-Ras4B. Previous studies have targeted H-Ras and not K-Ras4B as a target for the development of inhibitors. One recent report indicates that K-Ras4B can be geranylgeranylated in vitro, but with relatively low efficiency; its K.sub.m for GGTase I is 7 times higher than its K.sub.m for FTase (67). GGTase I CAAX-based inhibitors that can block geranylgeranylation processing have not been reported.
Recently, we have shown that a potent inhibitor of FTase disrupts K-Ras4B processing but only at very high concentrations that also inhibited the processing of geranylgeranylated proteins (66). This suggested that K-Ras4B may be geranylgeranylated, and that therefore inhibitors targeted at GGTase I would be effective in disrupting oncogenic K-Ras4B processing and signalling, and in treatment of cancers which were related to this form of Ras.